Nociceptin/orphanin FQ (N/OFQ) is an endogenous opioid-like peptide that produces marked effects on cardiovascular (hypotension, bradycardia, sympathoinhibition) and renal excretory (water diuresis) function in animals. Using selective ligands for the N/OFQ peptide receptor (NOP), we have obtained evidence that there exist separate pathways in the brain and periphery by which N/OFQ affects cardiovascular and renal function. As proposed in this application, the development of new ligands (peptide and non-peptide) selective for the NOP receptor, a pertussis toxin-sensitive G-protein receptor, will provide the basic pharmacological tools necessary to systematically study these tissue/system specific pathways. The hypothesis to be tested is that there exist separate central and peripheral NOP receptor pathways that affect cardiovascular and renal function, and that selective activation of the peripheral NOP receptor pathway with novel peptide and non-peptide ligands can evoke a free-water diuresis devoid of adverse cardiovascular/CNS effects. The investigations proposed in this amended application will test this hypothesis using a multidisciplinary approach that can be successfully achieved by the collaborative efforts of two established investigators in the N/OFQ research field, these being the P.I., and Dr. Domenico Regoli. This will entail the synthesis of novel NOP receptor ligands using peptide and non-peptide chemistry, which will be used to elucidate the biological actions, tissue distribution and signal transduction pathways of N/OFQ and NOP receptor ligands in the brain, periphery and kidneys. These studies will employ molecular, cellular and classical pharmacological approaches involving isolated organs, tissues and whole animals; and in vivo analysis of the cardiovascular and renal responses produced by these novel NOP receptor ligands. The use of genetically modified transgenic NOP receptor knockout mice (whole animal/tissue) will provide an innovative approach to understand the effects of N/OFQ and NOP ligands at each site. The pharmacological evaluation of N/OFQ and NOP receptor ligands in different in vitro bioassays and cell culture will explore the pharmacology of the NOP receptor system and the underlying signaling pathways by which NOP receptor ligands elicit diverse cardiovascular and renal responses. Finally, the potential for novel peripherally acting NOP ligands to produce a therapeutic water diuresis in states of vasopressin excess will be explored. It is anticipated that work in this area will provide new strategies for the treatment of different pathological states associated with fluid retention and/or hyponatremia/hypokalemia.